Canine pancreatic lipase

ABSTRACT

Isolated nucleic acid molecules having a nucleotide sequence encoding canine pancreatic lipase polypeptides, allelic variants and fragments thereof. Vectors and host cells containing the polynucleotide sequences and methods for expressing the polypeptides. Monoclonal antibodies that specifically binds to the canine pancreatic lipase polypeptides. Cell lines secreting the monoclonal antibodies. Methods for determining the presence or amount of canine pancreatic lipase in a biological sample. The methods include using the monoclonal antibodies to specifically bind to canine pancreatic lipase polypeptides. The method includes using standards of recombinant canine pancreatic lipase. Devices and kits for performing methods for detecting canine pancreatic lipase in biological samples.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional application of U.S. patent applicationSer. No. 11/107,086, filed Apr. 15, 2005, which claims the benefit ofU.S. provisional patent application Ser. No. 60/562,836 filed Apr. 16,2004 and U.S. provisional patent application Ser. No. 60/564,333 filedApr. 22, 2004.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention is related to the detection of pancreatic lipase. Morespecifically, the invention relates to pancreatic lipase polypeptides,polynucleotides encoding the polypeptides; antibodies specific for thepolypeptides, and method of using the polypeptides and antibodies todetect pancreatic lipase in biological samples.

2. Description of Related Art

Complete citations to the references described herein by author and dateare provided in the Bibliography section at the end of thespecification.

Lipases are water-soluble enzymes that hydrolyze water-insolublesubstrates into more polar lipolysis products (Petersen and Drablos1994). A plethora of lipases have been identified in microorganisms,plants, and animals (Lin et al., 1986; Jaeger et al., 1994; Petersen andDrablos, 1994; Mukherjee and Hills, 1994; Lawson et al., 1994). Lipasesshare a common triad of amino acids (serine, aspartic or glutamic acid,and histidine) in the active site, which is also shared with serineproteases (Svendsen, 1994). Another common feature of almost all lipasesare glycosylation site motifs (Antonian, 1988). Many lipases have beenshown to be related phylogenetically. The pancreatic lipase gene familyis a large gene family with 9 subfamilies (Petersen and Drablos, 1994;Carriere et al., 1997; Carriere et al., 1998; Hirata et al., 1999). Inaddition there are other groups of phylogenetically related lipases, andyet other lipases that do not belong to a defined gene family (Andersonand Sando, 1991).

The main function of lipases is the hydrolysis of lipids. A lipase isneeded whenever an apolar lipid needs to cross a biological membrane.Triglycerides are prime examples of apolar lipids. Thus lipase is neededin order for triglycerides to be absorbed from the intestinal tract.There are two digestive lipases in most vertebrate species, i.e., apreduodenal lipase and classical pancreatic lipase (Carriere et al.,1994). Preduodenal lipase has been shown to originate from a singletissue in all species examined to date (Moreau et al., 1988). Apharyngeal lipase was identified in cows and sheep, a lingual lipase inrats and mice, and a gastric lipase in human beings, monkeys, horses,pigs, guinea pigs, cats, and dogs (Moreau et al., 1988). No preduodenallipase could be identified in chickens (Moreau et al., 1988). In humanbeings and dogs it has been shown that gastric lipase contributessignificantly to the digestion of dietary triglycerides (Carriere etal., 1993a; Carriere et al., 1993b). However, pancreatic lipase (alsocalled classical pancreatic lipase) is the most important enzyme in thedigestion of dietary triglycerides (Carriere et al., 1991; Carriere etal., 1993a).

It has recently been shown by immunolocalization that pancreatic lipaseis only in pancreatic acinar cells in clinically healthy dogs,suggesting that classical pancreatic lipase may be an ideal marker forfunction and pathology of the exocrine pancreas (Steiner et al., 2002).This hypothesis has been confirmed in clinical studies that have shownthat the measurement of pancreatic lipase immunoreactivity in serum is aspecific marker for exocrine pancreatic function and also highlysensitive for pancreatitis in the dog (Steiner et al., 2001a; Steiner etal., 2001b; Steiner et al., 2001c).

Pancreatic lipase has an approximate molecular weight of 50 kilodaltons.The purification of classical pancreatic lipase has been reported inmany species (Vandermeers and Chroistophe, 1968; Rathelot et al., 1981;Bosc-Bieme et al., 1984; Gieseg et al., 1992; Mejdoub et al., 1994;Steiner and Williams, 2003).

Clinical symptoms of pancreatitis are non-specific and the disease canbe difficult to diagnose. Pancreatitis is associated with an increasedamount of digestive enzymes and zymogens leaking into the blood stream.One of these enzymes is pancreatic lipase. A number of assays have beendeveloped to detect the presence of lipase in serum by use of catalyticassays. However, these assays lack both sensitivity and specificity forpancreatitis in both human beings and dogs. Accordingly, what is neededis a simple and rapid method and device for sensitively and specificallydetecting pancreatic lipase.

SUMMARY OF THE INVENTION

In one aspect, the invention is directed to an isolated nucleic acidmolecule having a nucleotide sequence encoding canine pancreatic lipasepolypeptides, allelic variants or fragments thereof. The inventionincludes vectors and host cells containing the sequences, and methodsfor expressing the polypeptides.

The invention is also directed to monoclonal antibodies thatspecifically bind to the canine pancreatic lipase polypeptides. Theinvention further provides for a cell line secreting the monoclonalantibodies.

Another aspect of the invention is directed to methods for determiningthe presence or amount of canine pancreatic lipase in a biologicalsample. The method includes using the monoclonal antibodies tospecifically bind to canine pancreatic lipase polypeptides in thesample. The method includes using standards of recombinant caninepancreatic lipase.

Further aspects of the invention are directed to devices and kits forperforming methods for detecting canine pancreatic lipase in biologicalsamples.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the primer design for the identification andamplification of canine pancreatic lipase. Shown are a series ofdegenerate primers (1, 2, 3, 5) for 3′RACE (UPM—universal primer mix,Clontech) and nested PCR, as well as the primers used for 5′RACE (4, 6).The region of the previously published N-terminal amino acid sequence isshown.

FIG. 2 shows the 1.429 Kb canine pancreatic lipase gene, designated cPL1(SEQ ID NO: 2)

FIG. 3 shows the translated canine pancreatic lipase protein, designatedcPLP1 (SEQ ID NO. 3). The amino acid sequence was deduced from cDNAsequence analysis.

FIG. 4 shows a number of canine pancreatic lipase peptides [SEQ ID. NOs:10-52] which is generally a series of 20-mer peptides spanning SEQ IDNO. 3 in 10 amino acid sequence overlap.

FIG. 5 shows the purified, recombinant canine pancreatic lipasecontaining a 6×His tag. The protein can be identified in its purifiedform at approximately 55 kDa on either a Coomassie stained orHis-stained gel (A) or on Western blot using an anti-His monoclonalantibody or the 7E11 monoclonal antibody (B).

FIG. 6 depicts the antibody titers to cPLP1 in either DNA immunized mice(A) using a standard competition ELISA with the immune sera or inchickens (B) using the expressed recombinant protein as an immunogen.

FIG. 7 demonstrates the ability of the two monoclonal antibodies, 4G11and 7E11, to react with canine pancreatic lipase in canine serum.

FIG. 8 depicts the ability of monoclonal antibody 4G11 to inhibit theenzymatic activity of cPLP1.

FIG. 9 contains the ELISA data demonstrating that monoclonal antibodies4G11 and 7E11 do not compete with each other for binding to cPLP1.

FIG. 10 demonstrates the ability of monoclonal antibody 7E11 to competewith an anti-human pancreatic lipase antibody for binding to cPLP1.

FIG. 11 shows the results of an ELISA sandwich assay for caninepancreatic lipase using monoclonal antibodies 7E11 and 4G11.

DETAILED DESCRIPTION

As used herein, the singular forms “a,” “an”, and “the” include pluralreferents unless the context clearly dictates otherwise.

The N-terminal amino acid sequence from purified canine pancreaticlipase has been reported (Steiner and Williams, Biochimie 2002):

KEVCFPRLGCFSDDSPWAGIVERPL [SEQ ID NO:1]Based on this published amino acid sequence and on sequence similaritiesamong pancreatic lipases of other species, a series of degenerateprimers were designed and used for 3′RACE (Rapid Amplification of cDNAEnds) and nested PCR (FIG. 1) from which the complete 3′ end of the genewas obtained. Similarly, 5′RACE was used to obtain the 5′ end of thegene. The complete gene sequence (cDNA) and translated amino acidsequence is shown in FIGS. 2 and 3.

Accordingly, in one aspect the invention is directed to canine cDNAmolecules (e.g. designated herein cPL1, SEQ ID NO. 2), which encodecanine lipase proteins such as canine pancreatic lipase protein (e.g.designated herein as cPLP1, (SEQ ID NO. 3). cPLP1 protein, fragmentsthereof, derivatives thereof, and variants thereof are collectivelyreferred to herein as polypeptides of the invention or proteins of theinvention.

Accordingly, in one aspect, the invention is directed to isolatednucleic acid molecules encoding polypeptides of the invention orbiologically active portions thereof. The present invention providesnucleic acid sequences that encode protein molecules that have beenidentified as being members of the lipase family of proteins and arerelated to the pancreatic lipase subfamily (protein sequences areprovided in FIG. 3, transcript/cDNA sequences are provided in FIG. 2).The peptide sequences provided in FIG. 3, as well as the obviousvariants described herein, particularly allelic variants as identifiedherein and using the information in FIG. 3, will be referred herein asthe lipase peptides of the present invention, lipase peptides, orpeptides/proteins of the present invention.

The present invention provides isolated peptide and protein moleculesthat consist of, consist essentially of, or comprise the amino acidsequences of the lipase peptides disclosed in the FIG. 3, (encoded bythe nucleic acid molecule shown in FIG. 2,), as well as all obviousvariants of these peptides that are within the art to make and use. Someof these variants are described in detail below.

As used herein, a peptide is said to be “isolated” or “purified” when itis substantially free of cellular material or free of chemicalprecursors or other chemicals. The peptides of the present invention canbe purified to homogeneity or other degrees of purity. The level ofpurification will be based on the intended use. The critical feature isthat the preparation allows for the desired function of the peptide,even if in the presence of considerable amounts of other components (thefeatures of an isolated nucleic acid molecule is discussed below).

In some uses, “substantially free of cellular material” includespreparations of the peptide having less than about 30% (by dry weight)other proteins (i.e., contaminating protein), less than about 20% otherproteins, less than about 10% other proteins, or less than about 5%other proteins. When the peptide is recombinantly produced, it can alsobe substantially free of culture medium, i.e., culture medium representsless than about 20% of the volume of the protein preparation.

The language “substantially free of chemical precursors or otherchemicals” includes preparations of the peptide in which it is separatedfrom chemical precursors or other chemicals that are involved in itssynthesis. In one embodiment, the language “substantially free ofchemical precursors or other chemicals” includes preparations of thelipase peptide having less than about 30% (by dry weight) chemicalprecursors or other chemicals, less than about 20% chemical precursorsor other chemicals, less than about 10% chemical precursors or otherchemicals, or less than about 5% chemical precursors or other chemicals.

The isolated lipase peptide can be purified from cells that naturallyexpress it, purified from cells that have been altered to express it(recombinant), or synthesized using known protein synthesis methods. Forexample, a nucleic acid molecule encoding the lipase peptide is clonedinto an expression vector, the expression vector introduced into a hostcell and the protein expressed in the host cell. The protein can then beisolated from the cells by an appropriate purification scheme usingstandard protein purification techniques. Many of these techniques aredescribed in detail below.

Accordingly, the present invention provides proteins that consist of theamino acid sequences provided in FIG. 3 (SEQ ID NO:3), for example,proteins encoded by the transcript/cDNA nucleic acid sequences shown inFIG. 2 (SEQ ID NO:2). The amino acid sequence of such a protein isprovided in FIG. 3. A protein consists of an amino acid sequence whenthe amino acid sequence is the final amino acid sequence of the protein.

The present invention further provides proteins that consist essentiallyof the amino acid sequences provided in FIG. 3 (SEQ ID NO:3), forexample, proteins encoded by the transcript/cDNA nucleic acid sequencesshown in FIG. 2 (SEQ ID NO:2). A protein consists essentially of anamino acid sequence when such an amino acid sequence is present withonly a few additional amino acid residues, for example from about 1 toabout 100 or so additional residues, typically from 1 to about 20additional residues in the final protein.

The present invention further provides proteins that comprise the aminoacid sequences provided in FIG. 3 (SEQ ID NO:3), for example, proteinsencoded by the transcript/cDNA nucleic acid sequences shown in FIG. 2(SEQ ID NO:2). A protein comprises an amino acid sequence when the aminoacid sequence is at least part of the final amino acid sequence of theprotein. In such a fashion, the protein can be only the peptide or haveadditional amino acid molecules, such as amino acid residues (contiguousencoded sequence) that are naturally associated with it or heterologousamino acid residues/peptide sequences. Such a protein can have a fewadditional amino acid residues or can comprise several hundred or moreadditional amino acids. The preferred classes of proteins that arecomprised of the lipase peptides of the present invention are thenaturally occurring mature proteins. A brief description of how varioustypes of these proteins can be made/isolated is provided below.

The lipase peptides of the present invention can be attached toheterologous sequences to form chimeric or fusion proteins. Suchchimeric and fusion proteins comprise a lipase peptide operativelylinked to a heterologous protein having an amino acid sequence notsubstantially homologous to the lipase peptide. “Operatively linked”indicates that the lipase peptide and the heterologous protein are fusedin-frame. The heterologous protein can be fused to the N-terminus orC-terminus of the lipase peptide.

In some uses, the fusion protein does not affect the activity of thelipase peptide per se. For example, the fusion protein can include, butis not limited to, enzymatic fusion proteins, for examplebeta-galactosidase fusions, yeast two-hybrid GAL fusions, poly-Hisfusions, MYC-tagged, HI-tagged and Ig fusions. Such fusion proteins,particularly poly-His fusions, can facilitate the purification ofrecombinant lipase peptide. In certain host cells (e.g., mammalian hostcells), expression and/or secretion of a protein can be increased byusing a heterologous signal sequence.

A chimeric or fusion protein can be produced by standard recombinant DNAtechniques. For example, DNA fragments coding for the different proteinsequences are ligated together in-frame in accordance with conventionaltechniques. In another embodiment, the fusion gene can be synthesized byconventional techniques including automated DNA synthesizers.Alternatively, PCR amplification of gene fragments can be carried outusing anchor primers which give rise to complementary overhangs betweentwo consecutive gene fragments which can subsequently be annealed andre-amplified to generate a chimeric gene sequence (see Ausubel et al.,Current Protocols in Molecular Biology, 1992). Moreover, many expressionvectors are commercially available that already encode a fusion moiety(e.g., a GST protein). A lipase peptide-encoding nucleic acid can becloned into such an expression vector such that the fusion moiety islinked in-frame to the lipase peptide.

As mentioned above, the present invention also provides and enablesobvious variants of the amino acid sequence of the proteins of thepresent invention, such as naturally occurring mature forms of thepeptide, allelic/sequence variants of the peptides, non-naturallyoccurring recombinantly derived variants of the peptides, and paralogsof the peptides. Such variants can readily be generated using art-knowntechniques in the fields of recombinant nucleic acid technology andprotein biochemistry. It is understood, however, that variants excludeany amino acid sequences disclosed prior to the invention.

Such variants can readily be identified/made using molecular techniquesand the sequence information disclosed herein. Further, such variantscan readily be distinguished from other peptides based on sequenceand/or structural homology to the lipase peptides of the presentinvention. The degree of homology/identity present will be basedprimarily on whether the peptide is a functional variant ornon-functional variant, and the amount of divergence present in theparalog family.

To determine the percent identity of two amino acid sequences or twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Ina preferred embodiment, at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% ormore of the length of a reference sequence is aligned for comparisonpurposes. The amino acid residues or nucleotides at corresponding aminoacid positions or nucleotide positions are then compared. When aposition in the first sequence is occupied by the same amino acidresidue or nucleotide as the corresponding position in the secondsequence, then the molecules are identical at that position (as usedherein amino acid or nucleic acid “identity” is equivalent to amino acidor nucleic acid “homology”). The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which need to be introduced for optimal alignment of the twosequences.

The comparison of sequences and determination of percent identity andsimilarity between two sequences can be accomplished using amathematical algorithm. (Computational Molecular Biology, Lesk, A. M.,ed., Oxford University Press, New York, 1988; Biocomputing: Informaticsand Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of sequence Data, Part 1, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991). In a preferred embodiment, the percent identity betweentwo amino acid sequences is determined using the Needleman and Wunsch(J. Mol. Biol. (48):444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat http://www.gcg.com), using either a Blossom 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, thepercent identity between two nucleotide sequences is determined usingthe GAP program in the GCG software package (Devereux, J., et al,Nucleic Acids Res. 12(1):387 (1984)) (available at http://www.gcg.com),using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, thepercent identity between two amino acid or nucleotide sequences isdetermined using the algorithm of E. Myers and W. Miller (CABIOS,4:11-17 (1989)) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4.

The nucleic acid and protein sequences of the present invention canfurther be used as a “query sequence” to perform a search againstsequence databases to, for example, identify other family members orrelated sequences. Such searches can be performed using the NBLAST andXBLAST programs (version 2.0) of Altschul, et al. (J. Mol. Biol.215:403-10 (1990)). BLAST nucleotide searches can be performed with theNBLAST program, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to the nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to the proteinsof the invention. To obtain gapped alignments for comparison purposes,Gapped BLAST can be utilized as described in Altschul et al. (NucleicAcids Res. 25(17):3389-3402 (1997)). When utilizing BLAST and gappedBLAST programs, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used.

Full-length pre-processed forms, as well as mature processed forms, ofproteins that comprise one of the peptides of the present invention canreadily be identified as having complete sequence identity to one of thelipase peptides of the present invention as well as being encoded by thesame genetic locus as the lipase peptide provided herein.

Allelic variants of a lipase peptide can readily be identified as beinga canine protein having a high degree (significant) of sequencehomology/identity to at least a portion of the lipase peptide as well asbeing encoded by the same genetic locus as the lipase peptide providedherein. As used herein, two proteins (or a region of the proteins) havesignificant homology when the amino acid sequences are typically atleast about 70-80%, 80-90%, and more typically at least about 90-95% ormore homologous. A significantly homologous amino acid sequence,according to the present invention, will be encoded by a nucleic acidsequence that will hybridize to a lipase peptide encoding nucleic acidmolecule under stringent conditions as more fully described below.

Paralogs of a lipase peptide can readily be identified as having somedegree of significant sequence homology/identity to at least a portionof the lipase peptide, as being encoded by a gene from canines, and ashaving similar activity or function. Two proteins will typically beconsidered paralogs when the amino acid sequences are typically at leastabout 60%, or greater, and more typically at least about 70% or greaterhomology through a given region or domain. Such paralogs will be encodedby a nucleic acid sequence that will hybridize to a lipase peptideencoding nucleic acid molecule under moderate to stringent conditions asmore fully described below.

Non-naturally occurring variants of the lipase peptides of the presentinvention can readily be generated using recombinant techniques. Suchvariants include, but are not limited to deletions, additions andsubstitutions in the amino acid sequence of the lipase peptide. Forexample, one class of substitutions are conserved amino acidsubstitution. Such substitutions are those that substitute a given aminoacid in a lipase peptide by another amino acid of like characteristics.Typically seen as conservative substitutions are the replacements, onefor another, among the aliphatic amino acids Ala, Val, Leu, and Ile;interchange of the hydroxyl residues Ser and Thr; exchange of the acidicresidues Asp and Glu; substitution between the amide residues Asn andGln; exchange of the basic residues Lys and Arg; and replacements amongthe aromatic residues Phe and Tyr. Guidance concerning which amino acidchanges are likely to be phenotypically silent are found in Bowie etal., Science 247:1306-1310 (1990).

Variant lipase peptides can be fully functional or can lack function inone or more activities, e.g. ability to bind substrate, ability tohydrolyze substrate, etc. Fully functional variants typically containonly conservative variation or variation in non-critical residues or innon-critical regions. Functional variants can also contain substitutionof similar amino acids that result in no change or an insignificantchange in function. Alternatively, such substitutions may positively ornegatively affect function to some degree.

Amino acids that are essential for function can be identified by methodsknown in the art, such as site-directed mutagenesis or alanine-scanningmutagenesis (Cunningham et al., Science 244:1081-1085 (1989)),particularly using the results provided in FIG. 2. The latter procedureintroduces single alanine mutations at every residue in the molecule.The resulting mutant molecules are then tested for biological activitysuch as lipase activity or in assays such as an in vitro proliferativeactivity. Sites that are critical for binding partner/substrate bindingcan also be determined by structural analysis such as crystallization,nuclear magnetic resonance or photoaffinity labeling (Smith et al., J.Mol. Biol. 224:899-904 (1992); de Vos et al. Science 255:306-312(1992)).

The present invention further provides fragments of the lipase peptides,in addition to proteins and peptides that comprise and consist of suchfragments. In one aspect, the invention provides for the residuesidentified in FIG. 4. The fragments to which the invention pertains,however, are not to be construed as encompassing fragments that may bedisclosed publicly prior to the present invention.

As used herein, a fragment comprises at least 8, 10, 12, 14, 16, or morecontiguous amino acid residues from a lipase peptide. Such fragments canbe chosen based on the ability to retain one or more of the biologicalactivities of the lipase peptide or could be chosen for the ability toperform a function, e.g. bind a substrate or act as an immunogen.Particularly important fragments are biologically active fragments,peptides that are, for example, about 8 or more amino acids in length.Such fragments will typically comprise a domain or motif of the lipasepeptide, e.g., active site, a transmembrane domain or asubstrate-binding domain. Further, possible fragments include, but arenot limited to, domain or motif containing fragments, soluble peptidefragments, and fragments containing immunogenic structures. Predicteddomains and functional sites are readily identifiable by computerprograms well known and readily available to those of skill in the art(e.g., PROSITE analysis).

Polypeptides often contain amino acids other than the 20 amino acidscommonly referred to as the 20 naturally occurring amino acids. Further,many amino acids, including the terminal amino acids, may be modified bynatural processes, such as processing and other post-translationalmodifications, or by chemical modification techniques well known in theart. Common modifications that occur naturally in lipase peptides aredescribed in basic texts, detailed monographs, and the researchliterature, and they are well known to those of skill in the art (someof these features are identified in FIG. 3).

Known modifications include, but are not limited to, acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent crosslinks, formation of cystine, formation ofpyroglutamate, formylation, gamma carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination.

Such modifications are well known to those of skill in the art and havebeen described in great detail in the scientific literature. Severalparticularly common modifications, glycosylation, lipid attachment,sulfation, gamma-carboxylation of glutamic acid residues, hydroxylationand ADP-ribosylation, for instance, are described in most basic texts,such as Proteins—Structure and Molecular Properties, 2nd Ed., T. E.Creighton, W. H. Freeman and Company, New York (1993). Many detailedreviews are available on this subject, such as by Wold, F.,Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed.,Academic Press, New York 1-12 (1983); Seifter et al. (Meth. Enzymol.182: 626-646 (1990)) and Rattan et al. (Ann. N.Y. Acad. Sci. 663:48-62(1992)).

Accordingly, the lipase peptides of the present invention also encompassderivatives or analogs in which a substituted amino acid residue is notone encoded by the genetic code, in which a substituent group isincluded, in which the mature lipase peptide is fused with anothercompound, such as a compound to increase the half-life of the lipasepeptide (for example, polyethylene glycol), or in which the additionalamino acids are fused to the mature lipase peptide, such as a leader orsecretory sequence or a sequence for purification of the mature lipasepeptide or a pro-protein sequence.

Antibodies

The invention also provides antibodies that selectively bind to one ofthe peptides of the present invention, a protein comprising such apeptide, as well as variants and fragments thereof. As used herein, anantibody selectively binds a target peptide when it binds the targetpeptide and does not significantly bind to unrelated proteins. Anantibody is still considered to selectively bind a peptide even if italso binds to other proteins that are not substantially homologous withthe target peptide so long as such proteins share homology with afragment or domain of the peptide target of the antibody. In this case,it would be understood that antibody binding to the peptide is stillselective despite some degree of cross-reactivity.

As used herein, an antibody is defined in terms consistent with thatrecognized within the art: they are multi-subunit proteins produced by amammalian organism in response to an antigen challenge. The antibodiesof the present invention include polyclonal antibodies and monoclonalantibodies, as well as fragments of such antibodies, including, but notlimited to, Fab or F(ab′)₂, and Fv fragments.

Many methods are known for generating and/or identifying antibodies to agiven target peptide. Several such methods are described by Harlow,Antibodies, Cold Spring Harbor Press, (1989).

In general, to generate antibodies, an isolated peptide is used as animmunogen and is administered to a mammalian organism, such as a rat,rabbit or mouse. The full-length protein, an antigenic peptide fragmentor a fusion protein can be used. Particularly important fragments arethose covering functional domains, and domain of sequence homology ordivergence amongst the family, such as those that can readily beidentified using protein alignment methods and as presented in theFigures.

Antibodies are preferably prepared from regions or discrete fragments ofthe lipase proteins. Antibodies can be prepared from any region of thepeptide as described herein. However, preferred regions will includethose involved in function/activity and/or lipase/binding partnerinteraction.

An antigenic fragment will typically comprise at least 8 contiguousamino acid residues. The antigenic peptide can comprise, however, atleast 10, 12, 14, 16 or more amino acid residues. Such fragments can beselected on a physical property, such as fragments correspond to regionsthat are located on the surface of the protein, e.g., hydrophilicregions or can be selected based on sequence uniqueness.

In one aspect, the antibodies of the invention are monoclonal antibodiesproduced by a mouse myeloma cell line. This cell line can be made byfusing a mouse myeloma cell line with the spleen cells from mice thathave been injected with the complete canine pancreatic lipase protein,or antigenic portion thereof. As more completely described in theExamples below, two such cell lines have been deposited with theAmerican Type Culture Collection (ATCC), 10801 University Boulevard,Manassas Va., 20110-2209 on Mar. 31, 2005. These cell lines have beenassigned Patent Deposit Numbers PTA-6652 and PTA-6653. The deposits willbe maintained under the terms of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms. The depositsare provided as a convenience to those of skill in the art and are notan admission that the deposit is required under 35 U.S.C. § 112. Theantibodies secreted from the cell lines have been designated 4G11 and7E11.

Both antibodies bind to either the purified, native canine pancreaticlipase or the recombinant cPLP1. The antibodies do not compete for thesame epitope on cPLP1 and can be used in a sandwich ELISA. Bothantibodies bind native canine pancreatic lipase in canine serum.Antibody 4G11 partially inhibits the enzymatic activity of cPLP1,whereas 7E11 does not. Antibody 7E11 detects cPLP1 protein on Westernblots, whereas 4G11 does not. 7E11 competes with an anti-humanpancreatic lipase antibody for binding to cPLP1, whereas 4G11 does not.Antibody 4G11 appears to have a greater affinity for the cPLP1 than does7E11 based on the OD's obtained from a sandwich ELISA.

The antibodies can be used to isolate one of the proteins of the presentinvention by standard techniques, such as affinity chromatography orimmunoprecipitation. The antibodies can facilitate the purification ofthe natural protein from cells and recombinantly produced proteinexpressed in host cells. In addition, such antibodies are useful todetect the presence of one of the proteins of the present invention incells, tissues or fluids to determine the pattern of expression of theprotein among various tissues in an organism and over the course ofnormal development. Further, such antibodies can be used to detectprotein in situ, in vitro, or in a cell lysate or supernatant in orderto evaluate the abundance and pattern of expression. Also, suchantibodies can be used to assess abnormal tissue distribution orabnormal expression during development or progression of a biologicalcondition. Antibody detection of circulating fragments of the fulllength protein can be used to identify turnover.

Further, the antibodies can be used to assess expression in diseasestates such as in active stages of the disease or in an individual witha predisposition toward disease related to the protein's function. Whena disorder is caused by an inappropriate tissue distribution,developmental expression, level of expression of the protein, orexpressed/processed form, the antibody can be prepared against thenormal protein. If a disorder is characterized by a specific mutation inthe protein, antibodies specific for this mutant protein can be used toassay for the presence of the specific mutant protein.

Polynucleotides

The invention provides isolated polynucleotides encoding the caninepancreatic lipase. The term “lipase polynucleotide” or “lipase nucleicacid” refers to the sequence shown in SEQ ID NO:2. The term “lipasepolynucleotide” or “lipase nucleic acid” further includes variants andfragments of the lipase polynucleotide.

An “isolated” lipase nucleic acid is one that is separated from othernucleic acid present in the natural source of the lipase nucleic acid.Preferably, an “isolated” nucleic acid is free of sequences whichnaturally flank the lipase nucleic acid (i.e., sequences located at the5′ and 3′ ends of the nucleic acid) in the genomic DNA of the organismfrom which the nucleic acid is derived. However, there can be someflanking nucleotide sequences, for example up to about 5 KB. Theimportant point is that the lipase nucleic acid is isolated fromflanking sequences such that it can be subjected to the specificmanipulations described herein, such as recombinant expression,preparation of probes and primers, and other uses specific to the lipasenucleic acid sequences.

Moreover, an “isolated” nucleic acid molecule, such as a cDNA or RNAmolecule, can be substantially free of other cellular material, orculture medium when produced by recombinant techniques, or chemicalprecursors or other chemicals when chemically synthesized. However, thenucleic acid molecule can be fused to other coding or regulatorysequences and still be considered isolated.

In some instances, the isolated material will form part of a composition(for example, a crude extract containing other substances), buffersystem or reagent mix. In other circumstances, the material may bepurified to essential homogeneity, for example as determined by PAGE orcolumn chromatography such as HPLC. Preferably, an isolated nucleic acidcomprises at least about 50, 80 or 90% (on a molar basis) of allmacromolecular species present.

For example, recombinant DNA molecules contained in a vector areconsidered isolated. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells orpurified (partially or substantially) DNA molecules in solution.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofthe isolated DNA molecules of the present invention. Isolated nucleicacid molecules according to the present invention further include suchmolecules produced synthetically.

In some instances, the isolated material will form part of a composition(or example, a crude extract containing other substances), buffer systemor reagent mix. In other circumstances, the material may be purified toessential homogeneity, for example as determined by PAGE or columnchromatography such as HPLC. Preferably, an isolated nucleic acidcomprises at least about 50, 80 or 90% (on a molar basis) of allmacromolecular species present.

The lipase polynucleotides can encode the mature protein plus additionalamino or carboxyterminal amino acids, or amino acids interior to themature polypeptide (when the mature form has more than one polypeptidechain, for instance). Such sequences may play a role in processing of aprotein from precursor to a mature form, facilitate protein trafficking,prolong or shorten protein half-life or facilitate manipulation of aprotein for assay or production, among other things. As generally is thecase in situ, the additional amino acids may be processed away from themature protein by cellular enzymes.

The lipase polynucleotides include, but are not limited to, the sequenceencoding the mature polypeptide alone, the sequence encoding the maturepolypeptide and additional coding sequences, such as a leader orsecretory sequence (e.g., a pre-pro or pro-protein sequence), thesequence encoding the mature polypeptide, with or without the additionalcoding sequences, plus additional non-coding sequences, for exampleintrons and non-coding 5′ and 3′ sequences such as transcribed butnon-translated sequences that play a role in transcription, mRNAprocessing (including splicing and polyadenylation signals), ribosomebinding and stability of mRNA. In addition, the polynucleotide may befused to a marker sequence encoding, for example, a peptide thatfacilitates purification.

Lipase polynucleotides can be in the form of RNA, such as mRNA, or inthe form DNA, including cDNA and genomic DNA obtained by cloning orproduced by chemical synthetic techniques or by a combination thereof.The nucleic acid, especially DNA, can be double-stranded orsingle-stranded. Single-stranded nucleic acid can be the coding strand(sense strand) or the non-coding strand (opposite or anti-sense strand).

Lipase nucleic acid can comprise the nucleotide sequence shown in SEQ IDNO:2, corresponding to canine cDNA. In one embodiment, the lipasenucleic acid comprises only the coding region.

The invention further provides variant lipase polynucleotides, andfragments thereof, that differ from the nucleotide sequence shown in SEQID NO:2 due to degeneracy of the genetic code and thus encode the sameprotein as that encoded by the nucleotide sequence shown in SEQ ID NO:2.

The invention also provides lipase nucleic acid molecules encoding thevariant polypeptides described herein. Such polynucleotides may benaturally occurring, such as allelic variants (same locus), homologs(different locus) or may be constructed by recombinant DNA methods or bychemical synthesis. Such non-naturally occurring variants may be made bymutagenesis techniques, including those applied to polynucleotides,cells, or organisms. Accordingly, as discussed above, the variants cancontain nucleotide substitutions, deletions, inversions and insertions.

Typically, variants have a substantial identity with a nucleic acidmolecule of SEQ ID NO:2 and the complements thereof. Variation can occurin either or both the coding and non-coding regions. The variations canproduce both conservative and non-conservative amino acid substitutions.Homologs, and allelic variants can be identified using methods wellknown in the art. These variants comprise a nucleotide sequence encodinga lipase that is at least about 60-65%, 65-70%, typically at least about70-75%, more typically at least about 80-85%, and most typically atleast about 90-95% or more homologous to the nucleotide sequence shownin SEQ ID NO:2. Such nucleic acid molecules can readily be identified asbeing able to hybridize under stringent conditions, to the nucleotidesequence shown in SEQ ID NO:2 or a fragment of the sequence. It isunderstood that stringent hybridization does not indicate substantialhomology where it is due to general homology, such as poly A sequences,or sequences common to all or most proteins or all lipase enzymes.Moreover, it is understood that variants do not include any of thenucleic acid sequences that may have been disclosed prior to theinvention.

As used herein, the term “hybridizes under stringent conditions” isintended to describe conditions for hybridization and washing underwhich nucleotide sequences encoding a polypeptide at least about 60-65%homologous to each other typically remain hybridized to each other. Theconditions can be such that sequences at least about 65%, at least about70%, at least about 75%, at least about 80%, at least about 90%, atleast about 95% or more identical to each other remain hybridized to oneanother. Such stringent conditions are known to those skilled in the artand can be found in Current Protocols in Molecular Biology, John Wiley &Sons, N.Y. (1989), 6.3.1-6.3.6, incorporated by reference. One exampleof stringent hybridization conditions are hybridization in 6× sodiumchloride/sodium citrate (SSC) at about 45° C., followed by one or morewashes in 0.2×SSC, 0.1% SDS at 50-65° C. In another non-limitingexample, nucleic acid molecules are allowed to hybridize in 6× sodiumchloride/sodium citrate (SSC) at about 45° C., followed by one or morelow stringency washes in 0.2×SSC/0.1% SDS at room temperature, or by oneor more moderate stringency washes in 0.2×SSC/0.1% SDS at 42° C., orwashed in 0.2×SSC/0.1% SDS at 65° C. for high stringency. In oneembodiment, an isolated nucleic acid molecule that hybridizes understringent conditions to the sequence of SEQ ID NO:2 corresponds to anaturally-occurring nucleic acid molecule. As used herein, a“naturally-occurring” nucleic acid molecule refers to an RNA or DNAmolecule having a nucleotide sequence that occurs in nature (e.g.,encodes a natural protein).

As understood by those of ordinary skill, the exact conditions can bedetermined empirically and depend on ionic strength, temperature and theconcentration of destabilizing agents such as formamide or denaturingagents such as SDS. Other factors considered in determining the desiredhybridization conditions include the length of the nucleic acidsequences, base composition, percent mismatch between the hybridizingsequences and the frequency of occurrence of subsets of the sequenceswithin other non-identical sequences. Thus, equivalent conditions can bedetermined by varying one or more of these parameters while maintaininga similar degree of identity or similarity between the two nucleic acidmolecules.

The present invention also provides isolated nucleic acids that containa single or double stranded fragment or portion that hybridizes understringent conditions to the nucleotide sequence of SEQ ID NO:2 or thecomplement of SEQ ID NO:2. In one embodiment, the nucleic acid consistsof a portion of the nucleotide sequence of SEQ ID NO:2 or the complementof SEQ ID NO:2.

It is understood that isolated fragments include any contiguous sequencenot disclosed prior to the invention as well as sequences that aresubstantially the same and which are not disclosed. Accordingly, if afragment is disclosed prior to the present invention, that fragment isnot intended to be encompassed by the invention. When a sequence is notdisclosed prior to the present invention, an isolated nucleic acidfragment is at least about 6, preferably at least about 10, 13, 18, 20,23 or 25 nucleotides, and can be 30, 40, 50, 100, 200, 500 or morenucleotides in length. Longer fragments, for example, 30 or morenucleotides in length, which encode antigenic proteins or polypeptidesdescribed herein are useful.

Furthermore, the invention provides polynucleotides that comprise afragment of the full-length lipase polynucleotides. The fragment can besingle or double-stranded and can comprise DNA or RNA. The fragment canbe derived from either the coding or the non-coding sequence.

In another embodiment an isolated lipase nucleic acid encodes the entirecoding region. Other fragments include nucleotide sequences encoding theamino acid fragments shown in FIG. 4.

Thus, lipase nucleic acid fragments further include sequencescorresponding to the domains described herein, subregions alsodescribed, and specific functional sites. Lipase nucleic acid fragmentsalso include combinations of the domains, segments, and other functionalsites described above. A person of ordinary skill in the art would beaware of the many permutations that are possible.

Where the location of the domains or sites have been predicted bycomputer analysis, one of ordinary sill would appreciate that the aminoacid residues constituting these domains can vary depending on thecriteria used to define the domains. However, it is understood that alipase fragment includes any nucleic acid sequence that does not includethe entire gene. The invention also provides lipase nucleic acidfragments that encode epitope bearing regions of the lipase proteinsdescribed herein. Nucleic acid fragments, according to the presentinvention, are not to be construed as encompassing those fragments thatmay have been disclosed prior to the invention.

The nucleic acid fragments of the invention provide probes or primers inassays such as those described below. “Probes” are oligonucleotides thathybridize in a base-specific manner to a complementary strand of nucleicacid. Such probes include polypeptide nucleic acids, as described inNielsen et al. (1991) Science 254:1497-1500. Typically, a probecomprises a region of nucleotide sequence that hybridizes under highlystringent conditions to at least about 15, typically about 20-25, andmore typically about 40, 50 or 75 consecutive nucleotides of the nucleicacid sequence shown in SEQ ID NO:2 and the complements thereof. Moretypically, the probe further comprises a label, e.g., radioisotope,fluorescent compound, enzyme, or enzyme co-factor.

As used herein, the term “primer” refers to a single-strandedoligonucleotide which acts as a point of initiation of template-directedDNA synthesis using well-known methods (e.g., PCR, LCR) including, butnot limited to those described herein. The appropriate length of theprimer depends on the particular use, but typically ranges from about 15to 30 nucleotides. The term “primer site” refers to the area of thetarget DNA to which a primer hybridizes. The term “primer pair” refersto a set of primers including a 5′ (upstream) primer that hybridizeswith the 5′ end of the nucleic acid sequence to be amplified and a 3′(downstream) primer that hybridizes with the complement of the sequenceto be amplified.

Where the polynucleotides are used to assess lipase properties orfunctions, such as in the assays described herein, all or less than allof the entire cDNA can be useful. Assays specifically directed to lipasefunctions, such as assessing agonist or antagonist activity, encompassthe use of known fragments. Further, diagnostic methods for assessinglipase function can also be practiced with any fragment, including thosefragments that may have been known prior to the invention. Similarly, inmethods involving treatment of lipase dysfunction, all fragments areencompassed including those, which may have been known in the art.

The lipase polynucleotides are useful as a hybridization probe for cDNAand genomic DNA to isolate a full-length cDNA and genomic clonesencoding the polypeptide described in SEQ ID NO:3 and to isolate cDNAand genomic clones that correspond to variants producing the samepolypeptide shown in SEQ ID NO:3 or the other variants described herein.Variants can be isolated from the same tissue and organism from whichthe polypeptide shown in SEQ ID NO:3 were isolated, different tissuesfrom the same organism, or from different organisms. This method isuseful for isolating genes and cDNA that are developmentally-controlledand therefore may be expressed in the same tissue or different tissuesat different points in the development of an organism.

The probe can correspond to any sequence along the entire length of thegene encoding the lipase. Accordingly, it could be derived from 5′noncoding regions, the coding region, and 3′ noncoding regions. Thenucleic acid probe can be, for example, the full-length cDNA of SEQ IDNO:2 or a fragment thereof that is sufficient to specifically hybridizeunder stringent conditions to mRNA or DNA.

The lipase polynucleotides are also useful for constructing recombinantvectors. Such vectors include expression vectors that express a portionof, or all of, the lipase polypeptides. Vectors also include insertionvectors, used to integrate into another polynucleotide sequence, such asinto the cellular genome, to alter in situ expression of lipase genesand gene products. For example, an endogenous lipase coding sequence canbe replaced via homologous recombination with all or part of the codingregion containing one or more specifically introduced mutations. Thelipase polynucleotides are also useful for expressing antigenic portionsof the lipase proteins. The lipase polynucleotides are also useful formaking vectors that express part, or all, of the lipase polypeptides.The lipase polynucleotides are also useful as hybridization probes fordetermining the level of lipase nucleic acid expression. Accordingly,the probes can be used to detect the presence of, or to determine levelsof, lipase nucleic acid in cells, tissues, and in organisms. The nucleicacid whose level is determined can be DNA or RNA. Accordingly, probescorresponding to the polypeptides described herein can be used to assessgene copy number in a given cell, tissue, or organism. This isparticularly relevant in cases in which there has been an amplificationof the lipase genes.

Vectors/Host Cells

The invention also provides vectors containing the lipasepolynucleotides. The term “vector” refers to a vehicle, preferably anucleic acid molecule that can transport the lipase polynucleotides.When the vector is a nucleic acid molecule, the lipase polynucleotidesare covalently linked to the vector nucleic acid. With this aspect ofthe invention, the vector includes a plasmid, single or double strandedphage, a single or double stranded RNA or DNA viral vector, orartificial chromosome, such as a BAC, PAC, YAC, OR MAC.

A vector can be maintained in the host cell as an extrachromosomalelement where it replicates and produces additional copies of the lipasepolynucleotides. Alternatively, the vector may integrate into the hostcell genome and produce additional copies of the lipase polynucleotideswhen the host cell replicates.

The invention provides vectors for the maintenance (cloning vectors) orvectors for expression (expression vectors) of the lipasepolynucleotides. The vectors can function in procaryotic or eukaryoticcells or in both (shuttle vectors).

Expression vectors contain cis-acting regulatory regions that areoperably linked in the vector to the lipase polynucleotides such thattranscription of the polynucleotides is allowed in a host cell. Thepolynucleotides can be introduced into the host cell with a separatepolynucleotide capable of affecting transcription. Thus, the secondpolynucleotide may provide a trans-acting factor interacting with thecis-regulatory control region to allow transcription of the lipasepolynucleotides from the vector. Alternatively, a trans-acting factormay be supplied by the host cell. Finally, a trans-acting factor can beproduced from the vector itself.

It is understood, however, that in some embodiments, transcriptionand/or translation of the lipase polynucleotides can occur in acell-free system.

The regulatory sequence to which the polynucleotides described hereincan be operably linked include promoters for directing mRNAtranscription. These include, but are not limited to, the left promoterfrom bacteriophage lambda, the lac, TRP, and TAC promoters from E. coli,the early and late promoters from SV40, the CMV immediate earlypromoter, the adenovirus early and late promoters, and retroviruslong-terminal repeats.

In addition to control regions that promote transcription, expressionvectors may also include regions that modulate transcription, such asrepressor binding sites and enhancers. Examples include the SV40enhancer, the cytomegalovirus immediate early enhancer, polyomaenhancer, adenovirus enhancers, and retrovirus LTR enhancers.

In addition to containing sites for transcription initiation andcontrol, expression vectors can also contain sequences necessary fortranscription termination and, in the transcribed region a ribosomebinding site for translation. Other regulatory control elements forexpression include initiation and termination codons as well aspolyadenylation signals. The person of ordinary skill in the art wouldbe aware of the numerous regulatory sequences that are useful inexpression vectors. Such regulatory sequences are described, forexample, in Sambrook et al. (1989) Molecular Cloning: A LaboratoryManual 2nd. ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.).

A variety of expression vectors can be used to express a lipasepolynucleotide. Such vectors include chromosomal, episomal, andvirus-derived vectors, for example vectors derived from bacterialplasmids, from bacteriophage, from yeast episomes, from yeastchromosomal elements, including yeast artificial chromosomes, fromviruses such as baculoviruses, papovaviruses such as SV40, Vacciniaviruses, adenoviruses, poxviruses, pseudorabies viruses, andretroviruses. Vectors may also be derived from combinations of thesesources such as those derived from plasmid and bacteriophage geneticelements, e.g. cosmids and phagemids. Appropriate cloning and expressionvectors for prokaryotic and eukaryotic hosts are described in Sambrooket al. (1989) Molecular Cloning: A Laboratory Manual 2nd. ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

The regulatory sequence may provide constitutive expression in one ormore host cells (i.e. tissue specific) or may provide for inducibleexpression in one or more cell types such as by temperature, nutrientadditive, or exogenous factor such as a hormone or other ligand. Avariety of vectors providing for constitutive and inducible expressionin prokaryotic and eukaryotic hosts are well known to those of ordinaryskill in the art.

The lipase polynucleotides can be inserted into the vector nucleic acidby well-known methodology. Generally, the DNA sequence that willultimately be expressed is joined to an expression vector by cleavingthe DNA sequence and the expression vector with one or more restrictionenzymes and then ligating the fragments together. Procedures forrestriction enzyme digestion and ligation are well known to those ofordinary skill in the art.

The vector containing the appropriate polynucleotide can be introducedinto an appropriate host cell for propagation or expression usingwell-known techniques. Bacterial cells include, but are not limited to,E. coli, Streptomyces, and Salmonella typhimurium. Eukaryotic cellsinclude, but are not limited to, yeast, insect cells such as Drosophila,animal cells such as COS and CHO cells, and plant cells.

As described herein, it may be desirable to express the polypeptide as afusion protein. Accordingly, the invention provides fusion vectors thatallow for the production of the lipase polypeptides. Fusion vectors canincrease the expression of a recombinant protein, increase thesolubility of the recombinant protein, and aid in the purification ofthe protein by acting for example as a ligand for affinity purification.A proteolytic cleavage site may be introduced at the junction of thefusion moiety so that the desired polypeptide can ultimately beseparated from the fusion moiety. Proteolytic enzymes include, but arenot limited to, factor Xa, thrombin, and enterokinase. Typical fusionexpression vectors include pGEX (Smith et al. (1988) Gene 67:31-40),pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia,Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose Ebinding protein, or protein A, respectively, to the target recombinantprotein. Examples of suitable inducible non-fusion E. coli expressionvectors include pTrc (Amann et al. (1988) Gene 69:301-315) and pET 11d(Studier et al. (1990) Gene Expression Technology: Methods in Enzymology185:60-89).

Recombinant protein expression can be maximized in a host bacteria byproviding a genetic background wherein the host cell has an impairedcapacity to proteolytically cleave the recombinant protein. (Gottesman,S. (1990) Gene Expression Technology: Methods in Enzymology 185,Academic Press, San Diego, Calif. 119-128). Alternatively, the sequenceof the polynucleotide of interest can be altered to provide preferentialcodon usage for a specific host cell, for example E. coli. (Wada et al.(1992) Nucleic Acids Res. 20:2111-2118).

The lipase polynucleotides can also be expressed by expression vectorsthat are operative in yeast. Examples of vectors for expression in yeaste.g., S. cerevisiae include pYepSec1 (Baldari et al. (1987) EMBO J.6:229-234), pMFa (Kujan et al. (1982) Cell 30:933-943), pJRY88 (Schultzet al. (1987) Gene 54:113-123), and pYES2 (Invitrogen Corporation, SanDiego, Calif.).

The lipase polynucleotides can also be expressed in insect cells using,for example, baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., Sf9anc Sf21 cells) include the pAc series (Smith et al. (1983) Mol. Cell.Biol. 3:2156-2165) and the pVL series (Lucklow et al. (1989) Virology170:31-39).

In certain embodiments of the invention, the polynucleotides describedherein are expressed in mammalian cells using mammalian expressionvectors. Examples of mammalian expression vectors include pCDM8 (Seed,B. (1987) Nature 329:840), pMT2PC (Kauffman et al. (1987) EMBO J.6:187-195).

The expression vectors listed herein are provided by way of example onlyof the well-known vectors available to those of ordinary skill in theart that would be useful to express the lipase polynucleotides. Theperson of ordinary skill in the art would be aware of other vectorssuitable for maintenance propagation or expression of thepolynucleotides described herein. These are found for example inSambrook et al. (1989) Molecular Cloning: A Laboratory Manual 2nd, ed.,Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.

The invention also encompasses vectors in which the nucleic acidsequences described herein are cloned into the vector in reverseorientation, but operably linked to a regulatory sequence that permitstranscription of antisense RNA. Thus, an antisense transcript can beproduced to all, or to a portion, of the polynucleotide sequencesdescribed herein, including both coding and non-coding regions.Expression of this antisense RNA is subject to each of the parametersdescribed above in relation to expression of the sense RNA (regulatorysequences, constitutive or inducible expression, tissue-specificexpression).

The invention also relates to recombinant host cells containing thevectors described herein. Host cells therefore include prokaryoticcells, lower eukaryotic cells such as yeast, other eukaryotic cells suchas insect cells, and higher eukaryotic cells such as mammalian cells.

The recombinant host cells are prepared by introducing the vectorconstructs described herein into the cells by techniques readilyavailable to the person of ordinary skill in the art. These include, butare not limited to, calcium phosphate transfection,DEAE-dextran-mediated transfection, cationic lipid-mediatedtransfection, electroporation, transduction, infection, lipofection, andother techniques such as those found in Sambrook et al. (MolecularCloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory=Press, Cold Spring Harbor, N.Y.).

Host cells can contain more than one vector. Thus, different nucleotidesequences can be introduced on different vectors of the same cell.Similarly, the lipase polynucleotides can be introduced either alone orwith other polynucleotides that are not related to the lipasepolynucleotides such as those providing trans-acting factors forexpression vectors. When more than one vector is introduced into a cell,the vectors can be introduced independently, co-introduced or joined tothe lipase polynucleotide vector.

In the case of bacteriophage and viral vectors, these can be introducedinto cells as packaged or encapsulated virus by standard procedures forinfection and transduction. Viral vectors can be replication-competentor replication-defective. In the case in which viral replication isdefective, replication will occur in host cells providing functions thatcomplement the defects.

Vectors generally include selectable markers that enable the selectionof the subpopulation of cells that contain the recombinant vectorconstructs. The marker can be contained in the same vector that containsthe polynucleotides described herein or may be on a separate vector.Markers include tetracycline or ampicillin-resistance genes forprokaryotic host cells and dihydrofolate reductase or neomycinresistance for eukaryotic host cells. However, any marker that providesselection for a phenotypic trait will be effective.

While the mature proteins can be produced in bacteria, yeast, mammaliancells, and other cells under the control of the appropriate regulatorysequences, cell-free transcription and translation systems can also beused to produce these proteins using RNA derived from the DNA constructsdescribed herein.

Where secretion of the polypeptide is desired, appropriate secretionsignals are incorporated into the vector. The signal sequence can beendogenous to the lipase polypeptides or heterologous to thesepolypeptides.

Where the polypeptide is not secreted into the medium, the protein canbe isolated from the host cell by standard disruption procedures,including freeze thaw, sonication, mechanical disruption, use of lysingagents and the like. The polypeptide can then be recovered and purifiedby well-known purification methods including ammonium sulfateprecipitation, acid extraction, anion or cationic exchangechromatography, phosphocellulose chromatography, hydrophobic-interactionchromatography, affinity chromatography, hydroxylapatite chromatography,lectin chromatography, or high performance liquid chromatography.

It is also understood that depending upon the host cell in recombinantproduction of the polypeptides described herein, the polypeptides canhave various glycosylation patterns, depending upon the cell, or maybenon-glycosylated as when produced in bacteria. In addition, thepolypeptides may include an initial modified methionine in some cases asa result of a host-mediated process.

It is understood that “host cells” and “recombinant host cells” refernot only to the particular subject cell but also to the progeny orpotential progeny of such a cell. Because certain modifications mayoccur in succeeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term as usedherein.

Exemplary antigenic and enzymatic characteristics of cPLP1 which areexhibited by such polypeptides include lipase activity, ability to bindwith molecules with which cPLP1 is able to bind, and ability to induceproduction of antibody substances which bind specifically with anepitope which occurs at or near the surface of the cPLP1 protein. Thepolypeptides of the invention, or biologically active portions thereof,can be operably linked with a heterologous amino acid sequence to formfusion proteins. In addition, one or more polypeptides of the inventionor biologically active portions thereof can be incorporated intopharmaceutical compositions, which can optionally includepharmaceutically acceptable carriers. Such pharmaceutical compositionscan be used to treat or prevent one or more of the disorders identifiedherein. The invention encompasses antibody substances that specificallybind with a polypeptide of the invention including, for example, cPLP1protein and fragments thereof. Exemplary antibody substances that areincluded within the scope of the invention are monoclonal and polyclonalantibodies, antibody fragments, single-chain antibodies, free andcell-surface-bound antibodies, and T cell receptors. These antibodysubstances can be made, for example, by providing the polypeptide of theinvention to an immunocompetent vertebrate and thereafter harvestingblood or serum from the vertebrate. Antibody substances can,alternatively, be generated by screening a library of phage to identifyphage particles which display a subunit which binds with cPLP1 or anepitope thereof.

In another aspect, the invention provides methods for detecting activityor expression of a polypeptide of the invention in a biological sampleby contacting the biological sample with an agent capable of detectingsuch activity (e.g., a labeled substrate or another compound that can bedetected after being acted upon by an active polypeptide of theinvention), with an agent which binds specifically with a polypeptide ofthe invention (e.g., an antibody substance of the invention), or with anagent for detecting production of an RNA encoding a polypeptide of theinvention (e.g., a reverse transcriptase primer complementary to aportion of an mRNA encoding the polypeptide).

Detection of Canine Pancreatic Lipase

In one aspect, the invention is directed to an immunological method fordetecting the presence of an amount of canine pancreatic lipase in abiological sample. The invention provides a method, a device and a kitthat uses one or more canine lipase monoclonal antibodies. In anotheraspect, the method includes calibrators and standards comprising one ormore canine pancreatic lipase polypeptides.

“Binding specificity” or “specific binding” refers to the substantialrecognition of a first molecule for a second molecule, for example apolypeptide and a polyclonal or monoclonal antibody, or an antibodyfragment (e.g. a Fv, single chain Fv, Fab′, or F(ab′)2 fragment)specific for the polypeptide.

A “specific binding pair” is a set of two different molecules, where onemolecule has an area on its surface or in a cavity that specificallybinds to, and is therefore complementary to, an area on the othermolecule. “Specific binding partner” refers to one of these twocomplementarily binding molecules. “Specific binding pair” may refer toa ligand and a receptor, for example. In another example, the specificbinding pair might refer to an immunological pair, for example anantigen and antibody.

“Substantial binding” or “substantially bind” refer to an amount ofspecific binding or recognizing between molecules in an assay mixtureunder particular assay conditions. In its broadest aspect, substantialbinding relates to the difference between a first molecule'sincapability of binding or recognizing a second molecule, and the firstmolecules capability of binding or recognizing a third molecule, suchthat the difference is sufficient to allow a meaningful assay to beconducted distinguishing specific binding under a particular set ofassay conditions, which includes the relative concentrations of themolecules, and the time and temperature of an incubation. In anotheraspect, one molecule is substantially incapable of binding orrecognizing another molecule in a cross-reactivity sense where the firstmolecule exhibits a reactivity for a second molecule that is less than25%, preferably less than 10%, more preferably less than 5% of thereactivity exhibited toward a third molecule under a particular set ofassay conditions, which includes the relative concentration andincubation of the molecules. Specific binding can be tested using anumber of widely known methods, e.g, an immunohistochemical assay, anenzyme-linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), ora western blot assay.

A “biological sample” refers to a sample from an animal subjectincluding whole blood, serum, plasma, tissue, abdominal fluid (ascites),urine or other sample known or suspected to contain canine pancreaticlipase.

A “label” is any molecule that is bound (via covalent or non-covalentmeans, alone or encapsulated) to another molecule or solid support andthat is chosen for specific characteristics that allow detection of thelabeled molecule. Generally, labels are comprised of, but are notlimited to, the following types: particulate metal andmetal-derivatives, radioisotopes, catalytic or enzyme-based reactants,chromogenic substrates and chromophores, fluorescent andchemiluminescent molecules, and phosphors. The utilization of a labelproduces a signal that may be detected by means such as detection ofelectromagnetic radiation or direct visualization, and that canoptionally be measured.

The label employed in the current invention could be, but is not limitedto: alkaline phosphatase; glucose-6-phosphate dehydrogenase (“G6PDH”);horse radish peroxidase (HRP); chemiluminescers such as isoluminol,fluorescers such as fluorescein and rhodamine compounds; ribozymes; anddyes.

The label can directly produce a signal, and therefore additionalcomponents are not required to produce a signal. Alternatively, a labelmay need additional components, such as substrates or co-enzymes, inorder to produce a signal. The suitability and use of such labels usefulfor producing a signal are discussed in U.S. Pat. No. 6,489,309, andU.S. Pat. No. 5,185,243, which are incorporated by reference herein intheir entirety. For example, a label may be conjugated to the specificbinding partner in a non-covalent fashion. Alternatively, the label maybe conjugated to the specific binding partner covalently. U.S. Pat. No.3,817,837, and U.S. Pat. No. 3,996,345, which are incorporated byreference herein in their entirety, describe in detail example ofvarious ways that a label may be non-covalently or covalently conjugatedto the specific binding partner.

Solid phase means a porous or non-porous water insoluble material. Suchmaterials include a support or a surface such as the wall of a reactionvessel. The support can be hydrophilic or capable of being renderedhydrophilic and includes inorganic powders such as silica, magnesiumsulfate, and alumina; natural polymeric materials, particularlycellulosic materials and materials derived from cellulose, such as fibercontaining papers, e.g., filter paper, chromatographic paper, etc.;synthetic or modified naturally occurring polymers, such asnitrocellulose, cellulose acetate, poly (vinyl chloride),polyacrylamide, cross linked dextran, agarose, polyacrylate,polyethylene, polypropylene, poly(4-methylbutene), polystyrene,polymethacrylate, poly(ethylene terephthalate), nylon, poly(vinylbutyrate), etc.; either used by themselves or in conjunction with othermaterials; glass available as Bioglass, ceramics, metals, and the like.Natural or synthetic assemblies such as liposomes, phospholipidvesicles, and cells can also be employed.

Binding of sbp members to a support or surface may be accomplished bywell-known techniques, commonly available in the literature. See, forexample, “Immobilized Enzymes,” Ichiro Chibata, Halsted Press, New York(1978) and Cuatrecasas, J. Biol. Chem., 245:3059 (1970). The surface canhave any one of a number of shapes, such as strip, rod, particle,including bead, and the like. In one aspect, the polypeptides of theinvention include a N-terminal cysteine residue to assist in binding thepolypeptides to the solid phase.

The method of the invention can be optimized in many ways and one ofskill in the art could simultaneously adjust the sample dilutions,reagent concentrations, incubation temperatures and times used in themethod to accomplish detection of canine pancreatic lipase.

To be useful in the detection methods of the present invention, thepolypeptides are obtained in a substantially pure form, that is,typically from about 50% w/w or more purity, substantially free ofinterfering proteins and contaminants. Preferably, the polypeptides areisolated or synthesized in a purity of at least 80% w/w, and morepreferably, in at least about 95% w/w purity. Using conventional proteinpurification techniques, homogeneous polypeptide compositions of atleast about 99% w/w purity can be obtained. For example, the proteinsmay be purified by use of the antibodies described hereinafter using theimmunoabsorbant affinity columns described hereinabove.

The method of the invention may be accomplished using immunoassaytechniques well known to those of skill in the art, including, but notlimited to, using microplates and lateral flow devices. In oneembodiment, an antibody specific for canine pancreatic lipase protein isimmobilized on a solid support at a distinct location. Followingaddition of the sample, detection of protein-antibody complexes on thesolid support can be by any means known in the art. For example, U.S.Pat. No. 5,726,010, which is incorporated herein by reference in itsentirety, describes an example of a lateral flow device, the SNAP®immunoassay device (IDEXX Laboratories), useful in the presentinvention. In another aspect, the solid support is a well of amicrotiter plate.

Immobilization of one or more analyte capture reagents, e.g., antibodiesto canine pancreatic lipase, onto a device or solid support is performedso that an analyte capture reagent will not be washed away by thesample, diluent and/or wash procedures. One or more analyte capturereagents can be attached to a surface by physical adsorption (i.e.,without the use of chemical linkers) or by chemical binding (i.e., withthe use of chemical linkers). Chemical binding can generate strongerattachment of specific binding substances on a surface and providedefined orientation and conformation of the surface-bound molecules.

In another aspect, the invention includes one or more labeled specificbinding reagents that can be mixed with a test sample prior toapplication to a device for of the invention. In this case it is notnecessary to have labeled specific binding reagents deposited and driedon a specific binding reagent pad in the device. A labeled specificbinding reagent, whether added to a test sample or pre-deposited on thedevice, can be for example, a labeled canine pancreatic lipasemonoclonal antibody.

When the analyte capture reagent and the labeled specific bindingreagent are antibodies that specifically bind canine pancreatic lipase,the antibodies may be the same or different. In one aspect, theantibodies are chosen from 4G11 and 7E11 antibodies.

The detection method may include the use of a standard such as arecombinant canine pancreatic lipase polypeptide. The standard can bemixed with the monoclonal antibody or antibodies in the same manner asthe sample. The amount of binding between the monoclonal antibody orantibodies and the standard can be compared to the amount of binding ofthe antibodies to the protein in the sample. Accordingly, because theamount of canine pancreatic lipase in the standard is known, the amountof protein in the sample can be determined.

Any or all of the above embodiments can be provided as a kit. In oneparticular example, such a kit would include a device complete withspecific binding reagents (e.g., a non-immobilized labeled specificbinding reagent and an immobilized analyte capture reagent) and washreagent, as well as detector reagent and positive and negative controlreagents, if desired or appropriate. In addition, other additives can beincluded, such as stabilizers, buffers, and the like. The relativeamounts of the various reagents can be varied, to provide forconcentrations in solution of the reagents that substantially optimizethe sensitivity of the assay. Particularly, the reagents can be providedas dry powders, usually lyophilized, which on dissolution will providefor a reagent solution having the appropriate concentrations forcombining with a sample.

The device may also include a liquid reagent that transports unboundmaterial (e.g., unreacted fluid sample and unbound specific bindingreagents) away from the reaction zone (solid phase). A liquid reagentcan be a wash reagent and serve only to remove unbound material from thereaction zone, or it can include a detector reagent and serve to bothremove unbound material and facilitate analyte detection. For example,in the case of a specific binding reagent conjugated to an enzyme, thedetector reagent includes a substrate that produces a detectable signalupon reaction with the enzyme-antibody conjugate at the reactive zone.In the case of a labeled specific binding reagent conjugated to aradioactive, fluorescent, or light-absorbing molecule, the detectorreagent acts merely as a wash solution facilitating detection of complexformation at the reactive zone by washing away unbound labeled reagent.

Two or more liquid reagents can be present in a device, for example, adevice can comprise a liquid reagent that acts as a wash reagent and aliquid reagent that acts as a detector reagent and facilitates analytedetection.

A liquid reagent can further include a limited quantity of an“inhibitor”, i.e., a substance that blocks the development of thedetectable end product. A limited quantity is an amount of inhibitorsufficient to block end product development until most or all excess,unbound material is transported away from the second region, at whichtime detectable end product is produced.

In another aspect, the invention is directed to a kit for detectingcanine pancreatic lipase. For example the kit can include the devicedescribed above, along with the antibodies described herein. One or moreof the peptides of the invention can be included as a calibrator andcontrol. Such a kit can be supplied to detect a single protein orepitope or can be configured to detect one of a multitude of epitopes,such as in an antibody detection array. In one aspect, the kit includesa solid phase, such as a microtiter plate or lateral flow device, havingan immobilized antibody specific for canine pancreatic lipase, a reagentcomprising a second labeled antibody specific for canine pancreaticlipase, and reagents for use in detecting the label. The kit alsoincludes the appropriate packaging and instructions.

Other features and advantages of the invention will be apparent from thefollowing Examples. The following are provided for exemplificationpurposes only and are not intended to limit the scope of the inventiondescribed in broad terms above. All references cited in this disclosureare incorporated herein by reference.

EXAMPLE 1 Cloning and Characterization of the Canine Pancreatic Lipase(cPL1) Gene from Pancreatic Tissue

Based on the published N-terminal amino acid sequence of purified caninepancreatic lipase (Steiner and Williams, Biochimie 2002) (SEQ ID NO. 1)and sequence similarities among pancreatic lipases of other species, aseries of degenerate primers were designed and used for 3′RACE andnested PCR (FIG. 1). These primers targeted specific regions of thepancreatic lipase amino acid sequence which differentiate it from othermembers of the pancreatic lipase family, namely the pancreatic lipaserelated proteins. Total RNA was purified from canine pancreas usingTRIZOL® reagent (Invitrogen) and then reverse transcribed to cDNA usinga commercially available kit (SMART™ RACE cDNA Amplification Kit,Clontech). The 3′RACE reaction and nested PCR were successful inobtaining a 1.4 kb segment of the canine pancreatic lipase gene thatextended through to the appropriate stop codon. To complete the genesequence, cDNA for 5′ RACE was generated and specific primers within thecanine gene were designed for the RACE reaction as shown in FIG. 1. Thisamplification was successful in obtaining the complete 5′ end of thegene. The complete gene sequence (cDNA, SEQ ID NO: 2) and translatedamino acid sequence (SEQ ID NO: 3) are shown in FIGS. 2 and 3.

EXAMPLE 2 Expression of Canine Pancreatic Lipase

The gene for canine pancreatic lipase was amplified by PCR from caninepancreatic cDNA using a High Fidelity Taq according to themanufacturer's instructions (Roche) and ligated into a baculovirusexpression vector (pBlueBac4.5, Invitrogen). The reverse primer for thePCR contained the nucleotide sequence for a 6×His tag immediatelyfollowing the codon for the final amino acid of the protein. Thepurified vector was used for co-transfection with transfer DNA into Sf9insect cells (Invitrogen) using standard calcium phosphate techniques.Standard baculovirus protocols were followed to generate a high titerstocks of the recombinant virus for infection of Sf21 insect cells andprotein production (Invitrogen). Sf12 insect cells were grown inapproximately 500 ml serum-free EX-CELL™ 420 culture media (JRHBiosciences) to a concentration of 7-8×10⁸ cells and infected with 10 mlof virus stock, resulting in an MOI between 1.0-2.0. Following four daysof culture, activity of the recombinant cPLP1 protein was measured inthe culture supernatant using a standard lipase enzymatic assay (VITROS®Chemistry System, Ortho-Clinical Diagnostics).

The recombinant cPLP1 protein was purified from insect cell culturesupernatant following either standard protocols reported in theliterature (Thirstrup, K. et al. FEBS, 1993), or by means of the 6×Hisfusion tag metal chelate affinity chromatography (HISTRAP™ HP affinitycolumn, Amersham Biosciences). The purified protein was buffer exchangedinto phosphate buffered saline, pH7.2 using a standard desalting column(PD-10, Amersham Biosciences). The purified recombinant cPLP1 proteinwas shown to have lipase activity using a standard enzymatic assay(Vitros® Chemistry System).

As shown in FIG. 6A, the purified cPLP1 protein was also characterizedon SDS-PAGE gels using a Coomassie protein stain and an in-gel His-tagstain (Pierce) (FIG. 6A, lane 1). Pre-purification fractions are shownin lanes 2-4 with the molecular weight markers shown in lane 5. As shownin FIG. 6B, the purified cPLP1 protein could also be identified onWestern blots using an anti-His monoclonal antibody (1:200, anti-6Hisperoxidase, Roche) or the 7E11 monoclonal antibody (1:250, IDEXXLaboratories, Inc.).

EXAMPLE 3 Use of the Canine Pancreatic Lipase DNA and PolypeptideSequences for Immunization and Antibody Production

The gene for canine pancreatic lipase was amplified by PCR (HighFidelity Taq, Roche) from canine pancreatic cDNA and ligated into amammalian expression vector (pCMV-Tag4a, Stratagene) at the multiplecloning site. This vector may or may not be constructed with aC-terminal tag. The resulting vector was transiently transfected intoCOS7L cells using LIPOFECTAMINE™ Transfection Reagent (Invitrogen) toconfirm expression of the canine pancreatic lipase protein.

Purified vector DNA (MaxiPrep Kit, Qiagen) was used for DNA immunizationof mice according to published protocols (Ulmer, J. B. et al. Science,1993). Antibody titers from each individual mouse were evaluated twoweeks after the second immunization. A 96-well microtiter plate (Immulon2HB, Dynatech) was coated overnight at 4° C. with 10 μg/ml of ananti-human pancreatic lipase antibody (Fitzgerald #M410139a) inphosphate buffered saline (PBS, pH7.4). The plate was then blocked with3% BSA in 50 mM Tris (pH7.5) for 1 hour and washed 4 times in PBS-T(0.01M PBS with 0.05% Tween-20 (Sigma)). Sera from an unimmunized(negative control), immune (positive control), and two DNA vaccinatedmice were pre-incubated with a 1:1000 dilution of the recombinant,purified cPLP1 protein in antibody diluent (50 mM Tris (pH 7.2), 0.05%Tween-20, with both 50% fetal bovine serum and 10% mouse serum) for fiveminutes prior to adding it to the sandwich ELISA, thus creating acompetition format. The plate was incubated at room temperature for 1hour followed by 4 washes in PBS-T. The captured cPLP1 was detectedusing a 1:1000 dilution of a rabbit polyclonal antibody (Texas A&MUniversity, College Station, Tex.) and a 1:2500 dilution ofHRPO-conjugated goat anti-rabbit antibody (Jackson ImmunoResearch), inantibody diluent each for 1 hour at room temperature. The plate waswashed 6 times with PBS-T and developed with a TMB substrate (Moss,Inc.). As shown in FIG. 6A, reduction in signal (O.D.) relative to thenegative control indicates an antibody response to the cPLP1 antigen(FIG. 6A).

The purified, recombinant cPLP1 protein was also used as an immunogenfor antibody production in chickens. Two hens were immunized accordingto standard protocols familiar to those skilled in the art. After aseries of four injections, antibody titers were measured using asandwich ELISA with an anti-chicken HRP conjugate (1:2500, JacksonImmunoResearch) and the recombinant cPLP1, similar to the ELISAdescribed above. As shown in FIG. 6B, both hens developed reasonabletiters to cPLP1.

EXAMPLE 4 Use of Purified, Native Canine Pancreatic Lipase forImmunization and Antibody Production

Purified, native canine pancreatic lipase (Steiner and Williams,Biochimie. 2002 December; 84(12): 1245-53) was used to immunize Balb/Cmice using methods well known to those skilled in the art (seeAntibodies, a Laboratory Manual, by Harlow and Lane, Cold Spring HarborLaboratory Press, 1988, pp 53-135). Two mice were each immunized with˜63 ug of cPL using complete Freund's adjuvant, intraperitoneally (I.P.)on day 0. On day 25, using Freund's incomplete adjuvant, the mice wereboosted using the same procedure. On day 50, using Ribi adjuvant, themice were boosted using the same procedure.

On day 69, tail bleeds were taken and the anti-cPL titer was determinedusing an anti-cPL ELISA assay as described in Example 5, below.

On day 98 the mice were boosted subcutaneously (S.C.) with 30 ug ofnative cPL using Ribi adjuvant. On day 114 the mice were boosted usingan identical protocol. On day 123, tail bleeds were taken and theanti-cPL titer was determined using an anti-cPL ELISA assay. On day 143the mice were boosted intramuscularly in the hind leg with 10 ug ofnative cPL. On day 147 the spleens were harvested and fused with myelomacell line FO using methods well know to those skilled in the art (seeAntibodies, a Laboratory Manual, by Harlow and Lane, Cold Spring HarborLaboratory Press, 1988, pp 139-238).

EXAMPLE 5 ELISA for Canine Pancreatic Lipase

The method used for the initial screening of mouse tail bleeds isdescribed by Steiner et al. (Can. J. Vet. Res. 67:175-82). Briefly,canine pancreatic lipase was coated on 96 well microtitre plates at aconcentration of 0.3 ug/ml for 1 hour at 37° C. Plates were blocked withSuper Block (Pierce) for 1 hour and washed with PBS. Mouse sera sampleswere diluted 1:10 in PBS with 1% BSA and serially diluted across plate.Plates were incubated for 1 hour at 37° C., followed by 4 washes withPBS/0.05% tween20. Goat anti-mouse HRP conjugate (Jackson ImmunoResearch) diluted 1:3000 was used to detect bound antibodies. Plateswere developed with TMB reagent (Pierce).

EXAMPLE 6 Screening and Isolation of CaPL Monoclonal Antibodies

Hybridoma cell lines were grown as described in Example 4, andindividual monoclonal antibody producing clones were isolated using theprocess of limited dilution. A sandwich ELISA was developed to screenfor hybridoma's secreting antibodies specific for cPL.

Mouse monoclonal antibodies were captured from cell supernatants onImmulon 2 HB plates coated with donkey anti-mouse antibodies (JacksonImmuno Research) coated at a concentration of 10 ug/ml. Supernatentswere incubated on plates for 2 hours at room temperature (or overnightat 4° C.) to allow for capture to occur. Plates were then washed 6 timeswith PBS/0.1% Tween20 and incubated with canine pancreatic lipase (0.5ug/ml, 50 ul/well) for 1 hour and washed again. Rabbit anti-cPLpolyclonal antibody (Texas A&M University, College Station, Tex.)diluted 1:1000 in conjugate diluent (50 mM Tris (pH 7.2), 0.05%Tween-20, 50% fetal bovine serum) was added to wells and incubated for 1hour. Bound antibody was detected using a donkey anti-rabbit: HRPconjugate (Jackson Immuno Research) diluted 1:2500 in conjugate diluentPlates were washed 8 times before color development with TMB reagent.Color was allowed to develop for 5 minutes.

This method was used to identify monoclonal antibodies that boundspecifically to cPL. For example, two murine monoclonal antibodies wereisolated using this method, 4G11 and 7E11. These monoclonal antibodiesbind to cPL with suitable affinity for the development of a cPL ELISAassay. The cell lines secreting these antibodies have been depositedwith the ATCC, Manassas Va. on Mar. 30, 2005. Strain designations areCPL 7E11 clone 2/A5 and CPL 4G11/14D, bearing ATCC Patent DepositNumbers PTA-6653 and PTA 6652, respectively.

EXAMPLE 7 Characterization of Monoclonal Antibodies

Both of the identified monoclonal antibodies, 7E11 and 4G11, react withcPL in canine serum. Reactivity was demonstrated by using the ELISAformat described in this example and substituting two canine serumsamples (1:2 or 1:10 dilution in 3% BSA, 50 mM Tris (pH7.5)) for thecPLP1. Results are shown in FIG. 7.

Both of the identified monoclonal antibodies, 4G11 and 7E11, wereevaluated for their ability to interfere with the enzymatic activity ofthe cPLP1 in a lipase assay (VITROS® Chemistry System, Ortho-ClinicalDiagnostics). Hybridoma supernatant from either 4G11 and 7E11 was mixedwith filtered insect cell culture supernatant containing the cPLP1 togive a 1:10 dilution. Lipase activity was compared to a PBS control andan irrelevant hybridoma supernatant. Only the addition of hybridomasupernatant from 4G11 produced a reduction in enzymatic activity on thelipase assay (FIG. 8).

The identified monoclonal antibodies, 4G11 and 7E11, do not compete witheach other for binding to cPLP1. Using the ELISA protocol described inthis example, either 4G11 or 7E11 antibodies were captured on the platefrom the hybridoma supernatant. Recombinant cPLP1 (1:250 for 7E11 or1:1000 for 4G11) was diluted in the antibody diluent (see Example 3) inthe presence of either 10 μl 3% BSA, 10 μl hybridoma sup for 4G11, or 10μl hybridoma sup for 7E11, before being added to the microtiter plate. Areduction in signal (O.D.) was not observed for either monoclonalantibody when the antigen was pre-incubated with the alternatemonoclonal (FIG. 9).

Both identified monoclonal antibodies, 4G11 and 7E11, were tested fortheir ability to compete with a commercially available monoclonalantibody to human pancreateic lipase which we found to react with thecPLP1. An ELISA was performed as described in Example 3 where ananti-human pancreatic lipase antibody (Fitzgerald M410139a) was coatedonto microtiter plates. Wells were blocked with Tris-based Superblock(Pierce)+0.1% Tween-20. Following four washes in PBS-T, cPLP1 at a 1:500or 1:1000 dilution in antibody diluent (Example 3) was pre-incubated for10 minutes with either a 1:2, 1:5, or 1:10 dilution of 7E11 or 4G11,respectively prior to addition to the wells. Samples were incubated for1 hour followed by 5 washes in PBS-T. Detection with the polyclonalantibody to cPL was performed as described in Example 6. As shown inFIG. 10, the anti-human pancreatic lipase antibody does bind to cPLP1,and monoclonal antibody 7E11 competes with this antibody for binding ofcPLP1, but monoclonal antibody 4G11 does not.

When the reactivity of the purified monoclonal antibodies, 4G11 and7E11, are compared under equivalent concentrations, antibody 4G11 givesa greater O.D. (650 nm) reading for an equivalent concentration of cPLP1than does antibody 7E11. For instance, in an ELISA where each antibodyis coated on Immulon 2HB plates in PBS at 10 ug/ml overnight at 4° C.and processed with a 1:500 dilution of cPLP1 as described in Example 3,4G11 gives an O.D. reading of 1.747 vs. 7E11 which gives an O.D. readingof 1.383. Similarly, if the purified monoclonals are captured in anELISA as described in Example 6, a 1:4000 dilution of cPLP1 gives anO.D. reading for 4G11 of 1.010 vs. a 1:400 dilution of cPLP1 gives anO.D. reading for 7E11 of 1.140. This data suggests that these twomonoclonal antibodies have different binding affinities for the cPLP1antigen.

When compared biochemically, the two monoclonal antibodies, 4G11 and7E11, have different isoelectric focusing points. The pI for 4G11 is 6.7and the pI for 7E11 is 6.1.

EXAMPLE 8 Use of Antibodies Reactive to Canine Pancreatic Lipase

Antibodies recognizing the canine pancreatic lipase may be used inquantitative and non-quantitative assays for the detection of pancreaticlipase in canine serum or other biological samples. In one example, thecanine pancreatic lipase assay consists of an ELISA using the sandwichformat. In this format, monoclonal Anti-cPL (clone 7E11) is coated ontomicrotiter plate wells (Immulon 4 HBX plates; Thermo Electron Corp.;catalog number S25-343-04) at a concentration of approximately 5 ug/mL.The coating procedure is as follows: 7E11 monoclonal antibody is dilutedto 5 ug/mL in 10 mM phosphate buffered saline (PBS), pH 7.4. To eachwell, 100 uL of this coating solution is loaded and incubated at 4° C.for 8 hours. The coating solution is then aspirated and the plates arewashed in triplicate using 0.1M PBS/0.05% Tween 20. The plates are thenloaded with 200 uL per well of BSA-based blocking solution; the platesare incubated at 25° C. for 4 hours. The plates are aspirated and washedthree times with 0.1M PBS/0.05% Tween 20. Pancreatic lipase contained inthe serum sample or calibrator is captured by the solid-phase antibody.Calibrator preparation consists of diluting recombinant cPL antigen intoa BSA-based diluent to give calibrators at the ug/L level. Next,HRPO-conjugated monoclonal Anti-cPL (clone 4G11) is added to completethe sandwich. The HRPO-antibody conjugate is prepared using HRPO-SMCCand a disulfide reduced form of the antibody.

For this assay, the pancreatic lipase-containing calibrators and caninepatient samples are premixed in individual tubes along withHRPO-Conjugated mAb 4G11. The sample or calibrator to conjugate ratio is1:3 v/v. A conjugate dilution factor of 1:3000 is used in the assay. Nopremixture incubation time is required. The calibrator and samplepremixtures are then loaded into antibody-coated microtiter plate wells(100 μl), and incubated for one hour at 25° C. At the end of theincubation time, the plate is washed to remove unbound components. TMBsubstrate is added to the wells, and the plate is incubated for 5minutes at room temperature. The color reaction is stopped with theaddition of 1% SDS solution, and absorbance values are read at 650 nmusing a microtiter plate reader. Results using a mAB 7E11 and mAB 4G11sandwich and premixture protocol are shown in FIG. 11.

Alternatively, a protocol with no premixture may be followed. Thecalibrator or sample is loaded into the antibody-coated microtiter platewells, and incubated for one hour at 25° C. The plate is washed toremove unbound materials. The wells are then loaded with theHRPO-conjugated mAB 4G11 and incubated for one hour at 25° C. At the endof the incubation time, the plate is washed to remove unboundcomponents. TMB substrate is added to the wells, and the plate isincubated for 5 minutes at room temperature. The color reaction isstopped with the addition of 1% SDS solution, and absorbance values areread at 650 nm using a microtiter plate reader.

Although various specific embodiments of the invention have beendescribed herein, it is to be understood that the invention is notlimited to those precise embodiments and that various changes ormodifications can be affected therein by one skilled in the art withoutdeparting from the scope and spirit of the invention.

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1-32. (canceled) 33-67. (canceled)
 68. An isolated antibody for caninepancreatic lipase that specifically binds to a polypeptide having anamino acid sequence selected from the group consisting of (a) the aminoacid sequence of SEQ ID NO:3, (b) an amino acid sequence of an allelicvariant of SEQ ID NO:3, wherein the allelic variant is encoded by anucleic acid that hybridizes under stringent conditions to thecomplementary strand of a nucleic acid molecule of SEQ ID NO:2, and (c)an antigenic fragment of an amino acid sequence of SEQ ID NO:3.
 69. Theisolated antibody of claim 68 that is a monoclonal antibody.
 70. Theisolated antibody of claim 69, wherein the monoclonal antibody isselected from the group consisting of 4G11 and 7E11.
 71. The isolatedantibody of claim 69, wherein the monoclonal antibody is produced by acell line that is deposited with ATCC and has a patent deposit number ofPTA-6652 or PTA-6653.
 72. The isolated antibody of claim 71, wherein themonoclonal antibody is produced by a cell line that has a patent depositnumber of PTA-6652.
 73. The isolated antibody of claim 71, wherein themonoclonal antibody is produced by a cell line that has a patent depositnumber of PTA-6653.
 74. The isolated antibody of claim 71, wherein themonoclonal antibody is labeled.
 75. A monoclonal antibody specific forcanine pancreatic lipase polypeptide, wherein said monoclonal antibodyis produced by a cell line that is deposited with ATCC and has a patentdeposit number of PTA-6652 or PTA-6653.
 76. The monoclonal antibody ofclaim 75, wherein said monoclonal antibody is labeled.
 77. A cell linesecreting the monoclonal antibody of claim
 71. 78. The cell line ofclaim 75 wherein the cell line is deposited with ATCC and has a patentdeposit number selected from the group consisting of PTA-6652 andPTA-6653.
 79. A monoclonal antibody that competes with a monoclonalantibody produced by a cell line for binding with canine pancreaticlipase polypeptide, wherein said cell line is deposited with ATCC andhas a patent deposit number of PTA-6652 or PTA-6653.